Synthesis and in-vitro Evaluation of S-allyl Cysteine Ester - Caffeic Acid Amide Hybrids as Potential Anticancer Agents

We have synthesized a series of S-allyl cysteine ester-caffeic acid amide hybrids and evaluated them in order to determine their possible anticancer activity and selectivity in colorectal cancer, which is still one of the leading causes of morbidity and mortality worldwide. All compounds were tested against SW480 human colon adenocarcinoma cells and the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 6e, 9a, 9b, 9c, and 9e exhibited the highest effect on viability (IC50 SW480-48h= 0.18, 0.12, 0.12, 0.11, and 0.12 mM, respectively) and selectivity (SI = 10.3, 1.5, >83.33, >90.91 and >83.33, respectively) in a time- and concentration-dependent manner. Besides, our results were even better as regards lead compounds (S-allyl cysteine and caffeic acid) and the standard drug (5-FU). Additionally, these five compounds induced mitochondrial depolarization that could be related with an apoptotic process. Moreover, hybrids 6e, 9a, and 9e induced cell cycle arrest in G2/M phase, and compound 9c in S- phase, which suggests that these hybrid compounds could have also a cytostatic effect in SW480 cell line. The SAR analysis showed that hydroxyl groups increased the activity. Besides, there was not a clear relationship between the antitumor properties and the length of the alkyl chain. Since hybrid compounds were much more selective than the conventional drug (5-FU), this makes them promising candidates for further studies against colorectal cancer.


Introduction
Although colorectal cancer (CRC) can be prevented by healthy lifestyle habits this disease is still one of the leading causes of morbidity and mortality worldwide (1). In the most recent report of GLOBOCAN, the estimated burden of CRC until 2018 has exhibited an important increase through the years, being now the second most lethal cancer, preceded only by lung cancer. In addition, CRC is still the third most commonly diagnosed malignancy worldwide (2,3). Current chemotherapy for CRC involves multi-drug treatments such as FOLFIRI (folic acid/5-FU/irinotecan) and FOLFOX (5-FU/ leucovorin/oxaliplatin) which are composed of 5-fluorouracil as the backbone of treatment. Although these therapies are effective, there are many undesirable gastrointestinal and neurological side effects associated with these treatments, which many times result in dose limitations or cessation of the anti-cancer therapy (4,5). Given the toxicity of the current chemotherapy, it is necessary to discover new, more potent and selective agents for treating this disease. According to this, the studies dealing with hybrid compounds have been reported as a promising strategy (6), since these molecules may display a dual activity (7,8).
S-allyl cysteine (SAC), a naturally occurring water-soluble constituent of garlic, has exhibited antioxidant properties both in-vitro (9,10) and in-vivo (11)(12)(13). Furthermore, SAC displayed antiproliferative effects on neuroblastoma (14) and melanoma (15). Additionally, in prostate carcinoma cells this compound induced cell cycle arrest at the G0/G1 phases and cell apoptosis with decreased in Bcl-2 expression and increased expression of Bax and caspase 8 (16,17). Although this compound did not show growth-inhibitory effects in HCT15 colon cancer cells or in lung and skin carcinoma cell lines (18), its administration prior to 1,2-dimethylhydrazine (DMH) injection significantly inhibited colonic nuclear damage in female in a dose-dependent manner. These data show that the allyl group coupled to a single sulfur atom might play an important structural role in the inhibition of chemical toxicity and carcinogenesis induced with DMH in the colon (19).
On the other hand, caffeic acid and its derivatives display a broad spectrum of biological properties, among them, antioxidant (20,21), cytotoxic (22), pro-apoptotic, anti-inflammatory, and anti-angiogenic activity (23). Caffeic acid phenylethyl caffeate (CAPE, 1), Methyl caffeate 2 and Phenylethyl dimethyl caffeate 3 (Figure1) significantly inhibited cell growth and syntheses of RNA, DNA, and protein of HT-29 colon adenocarcinoma cells. Moreover, when CAPE was tested in CT26 colon adenocarcinoma cells, it exhibited a decrease in cell viability in a dose-dependent manner, without displaying significant influence on the growth of human umbilical vein epithelial cells (24). Additionally, high anti-proliferative activity was observed when 2´-propoxyl CAPE derivative 4 was tested against Hela and DU-145 human cancer cell lines (25). Furthermore, treatment with CAPE reduced the formation of aberrant crypt foci (ACF) and tumor in the rat colon (26). In addition, caffeic acid was found to be the least effective growth inhibitor of HT-29 cells when compared to its ester analogs, highligting the potency of the derivatives (27). In this sense, cyanoamide derivative 5 showed in-vitro inhibitory activities against human gastric carcinoma cell line BGC-823, human nasopharyngeal carcinoma cell line KB, and human hepatoma cell line BEL-7402 with IC 50 values of 5.6 µg/mL, 13.1 µg/mL, and activities against human gastric carcinoma cell line BGC-823, human nasopharyngeal carcinoma cell line KB, and human hepatoma cell line BEL-7402 with IC50 values of 5.6 µg/mL, 13.1 µg/mL, and 12.5 µg/mL, respectively (28). Finally, caffeic acid amide 6 and CAPE exhibited proapoptotic activity through activation of caspase-8 (29). There is an emerging strategy in medicinal chemistry and drug discovery research based on obtaining hybrid molecules that combine two or more structural fragments of the drugs that have a relevant pharmacological action (30,31). These hybrids may display dual activity but do not necessarily act on the same biological target (6,7). We have synthesized several S-allyl cysteine ester-caffeic acid amide S-allyl Cysteine Ester as Potential Anticancer Agents 12.5 µg/mL, respectively (28). Finally, caffeic acid amide 6 and CAPE exhibited proapoptotic activity through activation of caspase-8 (29).
There is an emerging strategy in medicinal chemistry and drug discovery research based on obtaining hybrid molecules that combine two or more structural fragments of the drugs that have a relevant pharmacological action (30,31). These hybrids may display dual activity but do not necessarily act on the same biological target (6,7). We have synthesized several S-allyl cysteine ester-caffeic acid amide hybrids ( Figure 2) and their effect on proliferation, mitochondrial membrane permeability, and cell cycle distibution was determined in order to identify posible therapeutic approaches for the treatment of colorectal cancer.

Chemical synthesis General remarks
Microwave reactions were carried out in a CEM Discover microwave reactor in sealed vessels (monowave, maximum power 300 W, temperature control by IR sensor, and fixed temperature). 1 H and 13 C NMR spectra were recorded on a Varian instrument operating at 300, 600 and 75, 125 MHz, respectively. The signals of the deuterated solvent (CDCl 3 or DMSO-D 6 ) were used as reference. Chemical shifts (δ) are expressed in ppm with the solvent peak as reference and TMS as an internal standard; coupling constants (J) are given in Hertz (Hz). Carbon atom types (C, CH, CH 2 , CH 3 ) were determined by using the DEPT pulse sequence. The signals were assigned using two-dimensional heteronuclear correlations (COSY, HSQC and HMBC). High resolution mass spectra were recorded using electrospray ionization mass spectrometry (ESI-MS). A QTOF Premier instrument with an orthogonal Z-spray-electrospray interface (Waters, Manchester, UK) was used operating in the W-mode. The drying and cone gas was nitrogen set to flow rates of 300 and 30 L/h, respectively. Methanol sample solutions (ca. 1 × 10 −5 M) were directly introduced into the ESI spectrometer at a flow rate of 10 µL/min. A capillary voltage of 3.5 kV was used in the positive scan mode, and the cone voltage set to Uc = 10 V. For accurate mass measurements, a 2 mg/L standard solution of leucine enkephalin was introduced via the lock spray needle at a cone voltage set to 85 V and a flow rate of 30 μL/min. IR spectra were recorded on a Spectrum RX I FT-IR system (Perkin-Elmer, Waltham, MA, USA) in KBr disks. Optical rotations were measured (Na-D line) at 25°C using a cell with 1dm path length on a Polartronic (Jasco model p-2000) polarimeter. Silica gel 60 (0.063-0.200 mesh, Merck, Whitehouse Station, NJ, USA) was used for column chromatography, and precoated silica gel plates (Merck 60 F254 0.2 mm) were used for thin layer chromatography (TLC). Monitoring of the reaction progress and product purification was carried out by TLC.

Procedure for the synthesis of S-Allylcysteine
(2): S-Cysteine hydrochloride (1 g, 6.34 mmol) was added to allyl bromide (1.15 g, 823 µL, 9.51 mmol) in 2M NH 4 OH (20 mL). The resulting hybrids ( Figure 2) and their effect on proliferation, mitochondrial membrane permeability, and cell cycle distibution was determined in order to identify posible therapeutic approaches for the treatment of colorectal cancer. Figure 2. Design of S-allyl cysteine ester-caffeic acid amide hybrids as anti-cancer agents.

General remarks
Microwave reactions were carried out in a CEM Discover microwave reactor in sealed vessels (monowave, maximum power 300 W, temperature control by IR sensor, and fixed temperature). 1 H and 13 C NMR spectra were recorded on a Varian instrument operating at 300, 600 and 75, 125 MHz, respectively. The signals of the deuterated solvent (CDCl3 or DMSO-D6) were used as reference.
Chemical shifts (δ) are expressed in ppm with the solvent peak as reference and TMS as an internal standard; coupling constants (J) are given in Hertz (Hz). Carbon atom types (C, CH, CH2, CH3) were determined by using the DEPT pulse sequence. The signals were assigned using two-dimensional heteronuclear correlations (COSY, HSQC and HMBC). High resolution mass spectra were recorded using electrospray ionization mass spectrometry (ESI-MS). A QTOF Premier instrument with an orthogonal Z-spray-electrospray interface (Waters, Manchester, UK) was used operating in the Wmode. The drying and cone gas was nitrogen set to flow rates of 300 and 30 L/h, respectively.
Methanol sample solutions (ca. 1 × 10 −5 M) were directly introduced into the ESI spectrometer at a flow rate of 10 µL/min. A capillary voltage of 3.5 kV was used in the positive scan mode, and the cone voltage set to Uc = 10 V. For accurate mass measurements, a 2 mg/L standard solution of leucine enkephalin was introduced via the lock spray needle at a cone voltage set to 85 V and a flow rate of 30 μL/min. IR spectra were recorded on a Spectrum RX I FT-IR system (Perkin-Elmer, Waltham, MA, mixture was stirred at room temperature for 20h. Then, the reaction mixture was concentrated to precipitate the product as a white solid. The solid was filtered, washed with ethanol (3 × 10 mL) and dried under reduced pressure, affording 818 mg (80%) of compound 2. This compound was used in the following step without further purification.

Procedure for the synthesis of S-Allylcysteine methyl ester (3a):
Thionyl chloride (442.6 mg, 3.72 mmol, 270 µL) was added over 5 min. to dry methanol (15 mL) cooled to -10 °C and the resulting solution was stored for a further 5 min. Then, S-allyl cysteine (500 mg, 3.1 mmol) was added and the mixture was stirred for 10 min. The resulting solution was stored at -10 °C for 2h, kept at room temperature for other 24 h, and then poured into ether (100 mL) and refrigerated for 2 h. The product (488 mg, 90%) separated as colorless needles, was removed by filtration.

General procedure for the synthesis of S-Allyl cysteine esters (3b-3e):
Thionyl chloride (3eq) was added over 5 min. to dry ethyl, propyl, butyl, or pentyl alcohol (15 mL) cooled to -10 °C and the resulting solution was stored for a further 5 min. Then, S-allyl cysteine (500 mg, 3.1 mmol) was added and the resulting mixture was stored at -10 °C for 2 h and the kept at room temperature for a further 24 h. Then the excess of alcohol was removed by distillation. The residue was purified by column chromatography over silica gel eluting with dichloromethane-methanol (95:5 ratio) to obtain S-allyl cysteine ethyl ester, S-allyl cysteine propyl ester, S-allyl cysteine butyl ester and S-allyl cysteine pentyl ester in 60% (352 mg), 71% (447 mg), 621% (418 mg) and 85% (609 mg) yields, respectively. Monitoring the reaction progress and product purification was carried out by TLC.

Procedure for desprotection of compounds 8a-8e
To a solution of compound 8 (1 mmol) in THF-H 2 O (1:1) (10 mL) was added KF (4 mmol) and the mixture was stirred for 12 h. Then an aqueous saturated solution of NH 4 Cl was added and the mixture was extracted with dichloromethane (3 × 10 mL).

Biological activity assays Cell lines and culture medium
Biological assays were performed using an adenocarcinoma colon cancer cell line (SW480) and non-malignant cells (CHO-K1). These were obtained from the European Collection of Authenticated Cell Cultures (ECACC, England) and maintained in Dulbecco's Modified Eagle Medium, supplemented with 10% heatinactivated (56 °C) horse serum, 1% penicillin/ streptomycin and 1% non-essential amino acids (Gibco Invitrogen, Carlsbad, USA). For all experiments, horse serum was reduced to 3%, and the medium was supplemented with 5 mg/ ml transferrin, 5 ng/mL selenium and 10 mg/ml insulin (ITS-defined medium; Gibco, Invitrogen, Carlsbad, USA) (32).

Cell Viability
The cell viability of the synthesized hybrids, lead, and reference compounds was evaluated through Sulforhodamine B (SRB) assay, a colorimetric test that is based on staining of total cellular protein of adherent cells. The cells were seeded to a final density of 20.000 cells/well in 96-well tissue culture plates and incubated at 37 °C in a humidified atmosphere at 5% CO 2 . All cultures were allowed to grow for 24 h and afterward they were treated with DMSO (dimethylsulfoxide; vehicle control 1%) or increasing concentrations (0.01-0.1 mM) of the synthesized hybrids, as well as SAC and caffeic acid (Lead compounds) and 5-fluorouracil (5-FU; the standard drug). After treatment, the cells were fixed with trichloroacetic acid (50% v/v) (MERCK) for a period of one h at 4 °C. The cell proteins were determined by staining with 0.4% (w/v) SRB (Sigma-Aldrich, United States), then they were washed with 1% acetic acid for the removal of unbound SRB and left for air-drying. Protein bound SRB was solubilized in 10 mM Tris-base and the absorbance was measured at 492 nm in a microplate reader (Mindray MR-96A) (33). All of the experiments were performed at least in quintuplicate.

Antiproliferative activity
Antiproliferative effect of the most active compounds was also tested through Sulforhodamine B (SRB) assay. Briefly, the cells were seeded to a final density of 2500 cells/well in 96-well tissue culture plates and incubated in the same conditions described for viability. The cultures were allowed to grow for 24 h and then were treated with increasing concentrations of the selected hybrids (0.1 -0.55 mM, ranges depended on the IC 50 -50% inhibitory concentration-values) or DMSO (vehicle control, 1%), for 0, 2, 4, 6, and 8 days. Culture media was replaced every 48 h. After each incubation time, the cells were fixed, stained, and read as previously described for this technique (32).

Measurement of Mitochondrial Membrane Potential (ΔΨm)
Mitochondrial membrane permeability changes were assessed through the fluorescent dye DiOC 6 (3,3'-dihexyloxacarbocyanine iodide, Thermo Fisher Scientific, Waltham, MA, USA), and propidium iodide (PI). The cells were seeded to a final density of 2.5 x 10 5 cells/well in 6-well tissue culture plates and were allowed to grow for 24 h. Then, they were treated with hybrids 6e, 9a, 9b, 9c, and 9e with its respective IC 50 (0.18, 0.12, 0.12, 0.11, and 0.12 mM, respectively), being harvested by scrapping at 48 h in the same culture mean, and stained with DiOC 6 and PI at room temperature for 30 min in darkness. The cells were collected to analyze 10,000 events by flow cytometry with excitation at 488 nm and detection of the emission with the green (530/15 nm) and the red (610/20 nm) filters. This method allowed us quantifying cells with depolarized mitochondrial membrane (34).

Cell cycle analysis
Cell cycle distribution was analyzed by labelling cells with propidium iodide (PI). Assays were carried out as described by Nicoletti et al. (1991). In brief, cells were seeded in 6-well tissue culture plates at a density of 2.5 x 10 5 cells/well, incubated at 37 °C in a 5% CO 2 atmosphere. The cultures were allowed to grow for 24 h and then were treated for 48 h with 1% DMSO (vehicle control) or hybrids 6e, 9a, 9b, 9c, and 9e with the IC 50 for each compound (0.18, 0.12, 0.12, 0.11, and 0.12mM, respectively). After the treatment, the cells were collected by scraping and the centrifuged cell pellet was resuspended with phosphate buffered saline (PBS). The cell suspension was fixed in 1.8 mL 70% ethanol at 4°C overnight, afterward, these were centrifuged, washed twice in PBS and resuspended in 300 µL of PBS containing 0.25 mg/mL RNAse (Type I-A, Sigma-Aldrich, Germany) and 0.1 mg/mL PI. Following the incubation in the dark at room temperature for 30 min, the PI fluorescence of 10,000 cells was analyzed using a FACS Canto II flow cytometer and the software BD FACS Diva 6.1.3. (BD Biosciences, San Jose). PI signal was analyzed with excitation at 488 nm, using a Sapphire laser, and fluorescence was detected at 610nm. The cell clumps were excluded with the PI-Area vs PI-Width signals.
The cell cycle model was fixed using the software FlowJo 7.6.2 (Ashland, OR, USA), applying the Dean-Jett-Fox model (34,35).

Statistical analysis
All experiments were performed at least three times. The data are reported as mean ± SE (standard error). Statistical differences between the control group (non-treated) and treated cells were evaluated by one-way ANOVA followed by the Dunnett′s test. Values with p ≤ 0.05 were considered significant. The data were analyzed with GraphPad Prism version 7.04 for Windows (Graph Pad Software, San Diego, California, USA).

Chemistry
The design of the compounds were based on lipophilic modifications. Allyl cysteine 2 was obtained, in 80% yield, via nucleophilic sustitution between cysteine 1 and allyl bromide (36). Reaction of 1 with the corresponding alcohol in the presence of thionyl chloride (37) afforded, after purification by crystallization or column chromatography, compounds 3a-3e in 60-90% yields. When these compounds were submitted to peptide type-coupling with 3,4-diacetoxycaffeic acid 5 (38) using HBTU as amide bond promoter (39), the compounds 6a-6e were achieved in 30-40% yields.
On the other hand, the phenolic hydroxyl groups of caffeic acid were protected as TBDMS ethers upon the reaction of reacting caffeic with TBDMSCl and imidazole in a solventfree reaction assisted by microwaves. In this conditions compound 7 was obtained in 50% yield (40). Then, the compound 7 was coupled with allylcysteine esters 3a-3e in the same conditions as above to afford amides 8a-8e in the yields ranging 50-60% (38). Finally, hybrids 9a-9e were obtained by deprotection from compounds 8 (yields 50-96%) (41). Obtention of hybrids 8 by deacetylation of compounds 6 (39, 42) was unsuccessful and a complex mixture was observed in all attempts carried out (Scheme 1).

Biological activity Effect of S-allyl cysteine ester-caffeic acid amide hybrids on SW480 and CHO-K1 cell viability
In order to assess their effect on the viability, the synthesized S-allyl cysteine ester-caffeic acid amide hybrids were evaluated against SW480 and CHO-K1 cell lines through the sulforhodamine B assay. As shown in Figure  3, the activity was time-and concentrationdependent, with a higher cytotoxic effect on SW480 cells in relation to CHO-K1 cells. Cytotoxicity was reported as 50% inhibitory concentration (IC 50 values).
All results regarding to the cytotoxic effect are summarized in Table 1. Among the compounds tested, hybrid 6b was the only one which did not exhibit activity neither at 24 h nor at 48 h after treatment. On the other hand, compounds 6a, 6c, and 6d exhibited good activity against the human colon adenocarcinoma SW480 cells, 24 h after treatment (IC50= 0.67, 1.02, and 2.79 mM, respectively); however, the effect decreased after 48 h as evidenced by the rise in the IC50 (>10 mM). Moreover, compounds 6e and 9a-9e showed the best activity against SW480 cells at 24 h after treatment, with IC50 values ranging from 0.10 to 0.93 mM, besides, the activity was maintained through the time since the IC50 values decreased 48 h after treatment with IC50 values in the range of 0.1-0.18 mM.
Additionally, their activity against the normal cell line was significantly lower compared to the reference drug, which gave these compounds great selectivity indexes (6e: 10.3; 9a: 1.5; 9b: >83.33 9c: >90.91 9d: >66.67; 9e: >83.33) after the 48 h treatment. It is important to highlight that compounds 9b-9e were much more selective than the lead compounds and the standard drug (5-FU). Compounds 3a and 3c, which correspond to allyl esters without hybridization, exhibited higher cytotoxicity against CHO-K1 thus displaying low selectivity indexes. The results regarding the activity of our hybrid compounds are in accordance with those reported by Herrera-R et al. (2018), which also found better activity and selectivity indices (SI) with some styrylcoumarin hybrids when tested in SW480 cells (32).

Figures 3.
Effect of S-allyl cysteine ester -cafeic acid amide hybrids on cell viability of SW480 and CHO-K1 cells, 48 h post-treatment with different concentrations (0.01-0.1mM). Cell viability was calculated using 100% viability of control. Data are presented as the mean ± SE of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). tested, hybrid 6b was the only one which did not exhibit activity neither at 24 h nor at 48 h after treatment. On the other hand, compounds 6a, 6c, and 6d exhibited good activity against the human colon adenocarcinoma SW480 cells, 24 h after treatment (IC 50 = 0.67, 1.02, and 2.79 mM, respectively); however, the effect decreased after 48 h as evidenced by the rise in the IC 50 (>10 mM). Moreover, compounds 6e and 9a-9e showed the best activity against SW480 cells at 24 h after treatment, with IC 50 values ranging from 0.10 to 0.93 mM, besides, the activity was maintained through the time since the IC 50 values decreased 48 h after treatment with IC 50 values in the range of 0.1-0.18 mM. Additionally, their activity against the normal cell line was significantly lower compared to the reference drug, which gave these compounds great selectivity indexes (6e: 10.3; 9a: 1.5; 9b: >83.33 9c: >90.91 9d: >66.67; 9e: >83.33) after the 48 h treatment. It is important to highlight that compounds 9b-9e were much more selective than the lead compounds and the standard drug (5-FU). Compounds 3a and 3c, which correspond to allyl esters without hybridization, exhibited higher cytotoxicity against CHO-K1 thus displaying low selectivity indexes. The results regarding the activity of our hybrid compounds are in accordance with those reported by Herrera-R et al. (2018), which also found better activity and selectivity indices (SI) with some styrylcoumarin hybrids when tested in SW480 cells (32).
In the structure-activity relationship (SAR) study, we noticed a synergistic action of the parent subunits when they are linked to form a single structure in the hybrid, as in the case of the compounds 6e and 9a-9e. Besides, allylcysteine esters such as 3a and 3c show a decreased activity, suggesting that the presence of the acid group is important within the mode of action that could involve the transference of a proton or formation of hydrogen bonds with a receptor (43). In addition, although higher selectivity indexes were achieved when the alkyl chain had two, three, and five carbon atoms (hybrids 9b, 9c and 9e, respectively), there was not a clear relationship between the antitumor activity and the length of the alkyl chain. On the other hand, hybrids with hydroxyl groups (9a-9e) showed better activity than acetylated derivatives (6a-6e), which is in agreement with the reports for several chalcones, coumarins, and caffeic acid esters (44)(45)(46), suggesting that this effect could be due to a better molecular recognition ability towards target bioreceptors upon hydrogen bond formation (43), oxidation processes across radical formation, and/or the ability of metal complexation (47).

Antiproliferative effect of S-allyl cysteine ester -caffeic acid amide hybrids on SW480 cells
The most active and selective compounds (6e, 9a-9e) were analized through a longer period of time in order to assess if they can display antiproliferative activity. After comparing each treatment with the control, the results indicated that the activity was time-and concentrationdependent. Among the results obtained, hybrids 6e, 9a-9c, and 9e displayed significant antiproliferative activity from day 2 onwards (p ≤ 0.05), even at the lowest concentrations evaluated, while compound 9d required a longer period of time (4 days) to exert activity against SW480 cells ( Figure 4A). Additionally, when observed with optical microscope, the cellular morphology of SW480 cells was severely perturbed, exhibiting changes in size and shape after treatment with S-allyl cysteine ester-caffeic acid amide hybrids, while the control displayed normal and healthy shape. Moreover, there was a clear decreased in number of the cells in comparison with the control, indicating either an increasing progression toward cell death or an arrest in cell cycle ( Figure 4B).

Changes in mitochondrial membrane potential (ΔΨm) induced by S-allyl cysteine ester -caffeic acid amide hybrids
The changes in Mitochondrial Membrane Potential (ΔΨm) could cause mitochondrial dysfunction, a process that is determinant in the execution of the cell death (48). Therefore, to assess the role of mitochondria in SW480 cells treated with our hybrids, the carbocyanine fluorescent dye DiOC6 was used. This dye accumulates in mitochondria due to its large negative membrane potential and it is released to the cytosol after a membrane depolarization (membrane with reduced ΔΨm), staining intracellular membranes (34, 49). According to the results (Figure 5), all hybrids caused a depolarization in the mitochondrial membrane as regards control, as observed in the decrease of the DiOC6 high population. Besides, in all cases, there was an increase in the field of the cells in latency and with loosing membrane polarization (DiOC6 low). Furthermore, there was not a great population with damaged membrane, which can be appreciated in the field of the cells stained with PI. This behavior was similar for all the evaluated compounds; however, hybrids 9a and 9e exhibited the highest activity in this cell line. A possible explanation for this could be related with the ability of hybrids 9a and 9e to induce cell death through a cross-talk between the extrinsic and intrinsic pathways of apoptosis via caspase-8 and Bid activation, which leads to mitochondrial membrane permeabilization (50).

S-allyl cysteine ester -caffeic acid amide hybrids induce cell cycle arrest on SW480 cells
Numerous studies have suggested that cancer progression involves the loss of checkpoint controls that regulate the passage through the cell cycle (51-54), which is critical in cancer pathogenesis and may affect the effectiveness of chemotherapy (17,55,56). Thus, we focused on determine whether hybrids 6e, 9a, 9b, 9c, and 9e could have any effect in the regulation of this process. The results obtained show that hybrids 6e, 9a, and 9e caused an arrest in the cell cycle in G 2 /M phase (22.7, 18.8 and 22.3%, respectively). In contrast, the proportion of the cells in G 0 /G 1 phase significantly decreased with regard to the control (p < 0.05). A similar reduction in G 0 /G 1 phase was observed with compound 9c (67.3%), along with an important increase in the S-phase of the cell cycle (23%). Additionally, compound 9b did not have any impact on the cell cycle. These findings complement our above findings, thus suggesting that our hybrids could have not only a cytotoxic effect but also a cytostatic activity ( Figure 6).

Conclusion
Our results demonstrate that some S-allyl cysteine ester-caffeic acid amide hybrids may display antiproliferative activity by inducing mitochondrial membrane depolarization and cell cycle arrest in SW480 cells, being even more active than the lead compounds. Besides, according to the results, the evaluated hybrids exhibited more selectivity than 5-FU, the conventional chemotherapeutic drug. The SAR analysis showed that hydroxyl groups increased the activity, besides, there was not a clear relationship between the antitumor properties and the length of the alkyl chain. All these findings make these hybrid compounds promising candidates for further antitumor studies.